Ion-channel based biological systems have not been extensively employed in current sensor technology because of the problems in reproducing a native-like microenvironment necessary for proper functioning. There are several problems to overcome in achieving this approach. First, a lipid bilayer must be formed upon a solid surface and the lipid bilayer should be sufficiently flexible and defect free such that ion-channel activity can actually be measured. Second, the lipid membrane must be in a physical state where biological molecules can function as if they are in a natural system. Third, an ionic medium needs to be entrapped between the membrane and the supporting electrode in order to facilitate the ion exchange through the ion-channels incorporated into the membrane.
A previous approach based on gramicidin-A pairs, by Cornell et al., Nature, vol. 387, pp. 580-583 (1997), has limitations related to a choice of ion-channel switching mechanism that is different from any of those present in cell membranes. Further, the preparation of the ionic reservoir structure between the membrane and the supporting electrode evidences high experimental difficulties. Their work has shown switched ion-channel sensors wherein the mode of transduction relies upon two antibodies for recognition, i.e., recognition opens or closes an ion channel by dragging gramacidin within the upper leaf away from gramacidin within the lower leaf that is anchored to the substrate surface thereby closing the channel. Another drawback to their approach is the need for a conjugation step (typically biotin-avidin) to attach the antibodies to molecules that anchor them to either the gramicidin or to a membrane spanning molecule that is also attached to the substrate surface.
Another previous approach has involved surface micro-patterning methods and this solid state approach has been useful for patterning supported phospholipid bilayer membranes. Such patterned phospholipid bilayer membranes have played an important role in the understanding, emulation, patterning and exploitation of selected functions of cell membranes for fundamental biophysical research and other biomedical and sensing technologies. Such micro-patterning of supported lipid membranes has been achieved by methods falling into two broad categories. The first is the use of pre-patterned substrate surfaces that present chemical and/or electrostatic barriers to membrane formation (see, e.g., Groves et al., Biophys. J., vol. 69, pp. 1972-1975 (1995)). The second is application of soft lithographic methods of patterned deposition, e.g., stamping, or removal, e.g., blotting, using polymeric stamps (see, e.g., Hovis et al., Langmuir, vol. 16, pp. 894-897 (2000).
Methods requiring substrate pre-patterning can depend upon the prior deposition of exogenous materials on the substrate surface and form single patterns. While methods based on polymer stamps can circumvent these issues, they require optimization of the physical contact, associated with contact pressure, and deformability of the polymer stamps. Moreover, some difficulties remain that are associated with achieving uniform contact for large-area patterning.
Morigaki et al. have recently described a photochemical method for micro-patterning of supported lipid membranes (see, Angew. Chemie. Int. Ed., vol. 40, pp. 172-174 (2001)). This method is based on a photolithographic polymerization of a diacetylene lipid that polymerizes with neighboring areas of lipids that are appropriately masked to avoid the polymerization process. The formation of a two-dimensional polymeric network makes the irradiated bilayer insoluble in organic solvents. By removing the monomeric lipids with an organic solvent, a two-dimensional corral structure is created for the incorporation of biologically relevant lipid bilayer membranes into the corrals. It was demonstrated that the newly incorporated bilayers retained their fluidity, whereas the polymerized lipid bilayer functioned as an effective barrier for the lateral diffusion of lipids. Despite these positive results in membrane micro-patterning, this method is limited by the need for photochemically polymerizable lipids.
Other approaches have relied upon single ion-channel proteins supported by a bilayer that spans a hole in a substrate such as a sheet of polytetrafluoroethylene (PTFE) or another hydrophobic polymer. Unfortunately, these approaches yield membranes with insufficient stability for most applications. Even small vibrations make them very unstable and easy to break. These black lipid membranes are particularly susceptible to disruption by vibrations or shock and typically can only be maintained for about 24 hours under the most favorable conditions. A similar approach has been described by Cheng et al., Langmuir, vol. 17, pp. 1240-1242 (2001), and involved a bilayer supported over a porous hydrophilic polymer.
Methods to create arrays of membranes would enable high-throughput screening of multiple targets against multiple drug-candidates. Arrays of membranes may be obtained by fabricating grids of titanium oxide on a glass substrate as titanium oxide resists the adsorption of lipids (Boxer, S. G. et al. Science 1997, 275, 651-653; and Boxer, S. G. et al. Langmuir 1998, 14, 3347-3350). Micro-pipeting techniques have been used to spatially address each corralled lipid-binding region (Cremer et al., J. Am. Chem. Soc., vol. 121, pp. 8130-8131 (1999)). However, these methods are cumbersome and require the fabrication of patterned surfaces. To make membrane arrays by printing membranes on unpatterned surfaces, it would be necessary to confine the membrane to the printed areas without lateral diffusion of the membrane molecules to the unprinted areas. Boxer et al. demonstrated that it was possible to pattern lipids on glass surfaces by microcontact printing using polydimethylsiloxane (PDMS) stamps “inked” with phosphatidylcholine. They attributed the lateral confinement of the lipids to the stamped regions, to the self-limiting expansion of phosphatidylcholine membranes to about 106% of the original printed areas (Hovis et al., Langmuir, vol. 16, pp. 894-897 (2000)). The methods used by Boxer et al., however, have certain limitations. First, Boxer et al. carried out the stamping of lipids on surfaces immersed under water. Second, lipids adsorbed on the bare-glass substrates used by Boxer et al. spontaneously desorbed when drawn through an air-water interface. In WO 01/20330, Cremer et al., propose the use of spatially addressed lipid bilayer arrays that remain submerged underwater to preserve the planar support structure. Such systems may not be practical for robust, high throughput, microarray based assays. Moreover, this approach could not be used to pattern addressable lipid bilayers onto conducting metal substrates.
Still other solutions to the existing problems have been sought.
The present inventors have now developed a simple light-directed method for patterning supported lipid membranes over large substrate areas in a non-contact manner and without the need for prior substrate patterning.